Ribonuclease (RNase, also known as RNA enzyme) is a large category of enzyme which includes dozens of types of enzymes capable of hydrolyzing RNA substrates into small molecular nucleic acids. According to the location of the cleavage site, these enzymes can be divided into two classes: endoribonuclease and exoribonuclease, belonging to a plurality of subclasses within EC 2.7 (phosphorylase) and EC 3.1 (hydrolase), respectively. RNases of different sources have different specificities and modes of action. For example, the catalysate of RNase T1 is mononucleotide, and oligonucleotide consisting of 3′-guanylic acid or carrying an end of 3′-guanylic acid, while the catalysate of RNase T2 is mononucleotide, and oligonucleotide consisting of 3′-adenylic acid or carrying an end of 3′-adenylic acid. Most RNases need divalent cations as cofactors (such as: Ca2+, Mg2+, and the like), so the activities thereof may be blocked by ethylenediaminetetraacetic acid (EDTA).
In laboratory studies, two main kinds of RNases are RNase A and RNase H. RNase H is an endoribonuclease which can specifically hydrolyze the phosphodiester bond on an RNA strand backbone complementary to a DNA strand, i.e., decompose the RNA strand in an RNA/DNA hybrid. However, this enzyme cannot digest single-stranded or double-stranded DNA. RNase A is another endonuclease which has been researched in details and has extensive applications. RNase A can hydrolyze RNA, whereas it does not function on DNA. RNase A effectively and specifically catalyzes the cleavage of phosphodiester bond on RNA strand backbone at 3′-end of pyrimidine nucleotide residues C and U to form oligonucleotides with 2′,3′-cyclic phosphate derivatives, thus it can be used to remove RNA contaminant in DNA preparations.
In order to detect the activity or content of RNase in a sample, the researchers have established many technical solutions on the basis of analyzing substrate degradation. These solutions can be roughly classified into two categories: one uses heterogeneous RNA molecules as substrates, and the other uses artificially synthesized RNA molecules as substrates, oligoribonucleotides for example. Normally, the detection technique using artificially synthesized oligoribonuclec acids as substrates has higher sensitivity and higher specificity. Among these solutions, numerous detection techniques are artfully applied, including direct staining method, spectrophotometric method, colorimetric detection, cascade chromogenic method, isotope tracer technique, fluorescence polarization technique and fluorescence quenching technique.
The selection of detection means to a large extent decides the sensitivity and application scope of analytical techniques. For example, in order to detect whether an experimental reagent is contaminated by RNase, a very sensitive detection technique is needed to determine the existence of a minute amount of RNase in the experimental reagent, thereby avoiding degradation of the experimental sample. If the detection means is not sensitive enough, it will be unable to detect the existence of RNase with a minute amount but is sufficient to influence the experimental result and will directly result in failure of the experiment. On the contrary, if the detection means is too sensitive, it will exclude the reagents containing an extremely minute amount of RNase which does not suffice to influence the experimental result. Although such high sensitivity can guarantee accuracy of the experimental result, it meanwhile will raise difficulty and cost of the experiment. In actual practice, it is ideal that the detection sensitivity is in a range of 1-100 picogram/ml (pg/ml) RNase A, but the sensitivity of commercial detection reagents is in a range of 10-100 pg/ml RNase A at present. On the other hand, as the monitoring of RNase is a routine laboratory operation, the detection technique is required to have relatively high stability and be easy to operate.
Earlier RNase activity detection was established on Kunitz technique (Kunitz M., A spectrophotometric method for the measurement of ribonuclease activity. J. Biol. Chem., 1946, 164:563-568). In this technique, a heterogeneous RNA sample was added into a test sample, the change in absorbance value of the RNA sample at 300 nm was detected by spectrophotometric method, and the activity of RNase in the sample was calculated. Shortly afterwards, Oshima optimized this technique (Oshima T, Uenishi N and Imahori K. Simple assay methods for ribonuclease T1, T2, and nuclease PI. Anal. Biochem., 1976, 71:632-634). Generally, the sensitivity of this detection solution is not high and does not meet the requirements for RNase activity detection.
In another RNase activity detection solution, firstly polyacrylamide gel was used to separate the sample (Wilson C W. A rapid staining technique for detection of RNase after polyacrylamide gel electrophoresis. Anal. Biochem., 1969, 31:506-511). By bringing the RNase sample separated by the gel into contact with a heterogeneous RNA substrate followed by RNA staining, the degradation condition of the RNA substrate was analyzed, and then RNase activity was detected. Although this solution is simple in terms of concept, the implementation thereof is rather time-consuming, and its sensitivity is not high (only RNase I of one unit or more can be detected). In the modified version of this solution, by improving the gel separation technique, Karpetsky raised the detection sensitivity to a degree capable of detecting RNase A with an amount of no more than 100 pg (Karpetsky T P, Davie G E, Shriven K K and Levy C C. Use of polynucleotide/polyacrylamide-gel electrophoresis as a sensitive technique for the detection and comparison of ribonuclease activities. Biochem. J., 1980, 189: 277-284). Even so, the modified solution does not effectively shorten the detection time. As a result, the solution still does not meet the need of experimental tests.
Another RNase activity detection solution was established by Egly and Kempf (Egly J M and Kempf J. Detection and estimation of very low ribonuclease activities in biological fluids. FEBS Letters, 1976, 63:250-254). By using RNase to degrade insoluble substrate and analyzing the nucleotides labeled by 125Iodine generated from degradation, the detection sensitivity of this solution may reach 0.01 pg/ml, which is suitable for applied in routine quality control and detection. However, as this technique adopts hazardous radioisotope, it is not suitable for routine use of ordinary laboratories or industries and its applicable scope is greatly restricted.
Another RNase activity detection technique was established by Wagner (Wagner A P, Iordachel M C and Wagner L P. A simple spectrophotometric method for the measurement of ribonuclease activity in biological fluids. J. Biochem. Biophys. Methods, 1983, 8:291-297). Through binding RNA with Pyronine-Y, the maximum absorbance peak value of RNA was raised to 572 nm, and the degradation of RNA by RNase in the sample resulted in linear reduction of this absorbance. The sensitivity of this detection technique is around 2 ng/ml RNase A, not suitable for the detection of RNase activity.
Another RNase detection technique was established by Greiner-Stoeffele (Greiner-Stoeffele T, Grunow M and Hahn U. A general ribonuclease assay using methylene blue. Anal. Biochem., 1996, 240:24-28). The dye methylene blue was intercalated into high-molecular-weight RNA substrate to form a dye-RNA complex, and the degradation of the RNA substrate by RNase in the sample released the dye from the complex, resulting in reduction of the absorbance at 688 nm. This solution also has the problem of low sensitivity, with a lower detection limit of 25 ng/ml only, not meeting the need of detecting RNase activity in conventional samples.
Another RNase detection technique was established by Karn (Karn R C, Crisp M, Yount E A and Hodes M E. A positive zymogram method for ribonuclease. Anal. Biochem., 1979, 96:464-468). In this solution, upon utilizing the degradation of a synthesized substrate by RNase A, a series of cascade reactions occurred and finally a detectable blue substance was formed. This detection solution may detect 0.066 unit of RNase A (˜100 ng), which does not meet the need of common RNase detection, either.
Another RNase detection technique was established by Witmer (Witmer M R, Falcomer C M, Weiner M P, Kay M S, Begley T P, Ganem B and Scheraga H A. U-3′-BCIP: a chromogenic substrate for the detection of RNase A in recombinant DNA expression systems. Nucleic Acids Res., 1991, 19:1-4). The main feature of this technique was the synthesis of an RNase substrate U-3′-BCIP which would release a reporter group under the action of RNase. The release of the reporter group was detected by spectrophotometer at 650 nm, thereby calculating the activity and content of RNase in the sample. This solution is simple and convenient, but it is still not sensitive enough.
Fluorescence quenching technique is a research technique widely applied in different fields of life science. For example, it has been used to detect the activity of protease (Yaron A, Carmel A and Katchalski-Katzir E. Intramolecularly quenched fluorogenic substrates for hydrolytic enzymes. Anal. Biochem., 95:228-235, 1979), detect the activity of DNA restriction endonuclease (Ghosh S S, Eis P S, Blumeyer K, Fearon K and Millar D P. Real time kinetics of restriction endonuclease cleavage monitored by fluorescence resonance energy transfer. Nucleic Acids Res., 1994, 22:3155-3159), detect the 5′ exonuclease activity of DNA polymerase (Gelfand D H, Holland P M, Saiki R K and Watson R M. Homogeneous assay system using the nuclease activity of a nucleic acid polymerase. U.S. Pat. No. 5,210,015, 1993), detect specific nucleic acid sequences (Gelfand D H, Holland P M, Saiki R K and Watson R M. Homogeneous assay system using the nuclease activity of a nucleic acid polymerase. U.S. Pat. No. 5,210,015, 1993; Tyagi S, Kramer F R and Lizardi P M. Detectably labeled dual conformation oligonucleotide probes, assays and kits. U.S. Pat. No. 5,925,517, 1999; Livak K J, Flood S J A, Marmaro J and Mullah K B. Hybridization assay using self-quenching fluorescence probe. U.S. Pat. No. 5,876,930, 1999; Nazarenko I A, Bhatnagar S K, Winn-Deen E S and Hohman R J. Nucleic acid amplification oligonucleotides with molecular energy transfer labels and methods based thereon. U.S. Pat. No. 5,866,336, 1999; Nadeau J G, Pitner B, Linn C P and Schram J L. Detection of nucleic acids by fluorescence quenching. U.S. Pat. No. 5,958,700, 1999), and detect immunoreaction (Maggio E T. Chemically induced fluorescence immunoassay. U.S. Pat. No. 4,220,450, 1980). In a solution which applied fluorescence resonance energy transfer technique to detect the activity of hammerhead ribozyme, a synthetic oligoribonucleic acid substrate was used, wherein one end of the oligoribonucleic acid substrate was attached to a FAM fluorophore and the other end was attached to a TAMRA quencher (Hanne A, Ramanujam M V, Rucker R and Krupp G. Fluorescence resonance energy transfer (FRET) to follow ribozyme reactions in real time. Nucleosides and Nucleotides. 1998, 17:1835-1850).
In another technical solution, Zelenko et al synthesized a dinucleotide substrate, wherein one end thereof was attached to a fluorophore (O-aminobenzoic acid) and the other end was attached to a quencher (2,4-dinitroaniline) (Zelenko O, Neumann U, Brill W, Pieles U, Moser H E and Hofsteenge J. A novel fluorogenic substrate for ribonucleases: synthesis and enzymatic characterization. Nucleic Acids Res., 1994, 22:2731-2739). Cleavage of this substrate by RNase A allowed the fluorophore to be separated from the quencher, leading to an increase in the fluorescence reading of the reaction system. This solution is designed to investigate enzymatic kinetics of RNase A, which has certain limitations in detection sensitivity and is not suitable for the detection of RNase activity of conventional samples, either.
In another solution, James designed a 9-mer chimeric oligonucleotide for studying the enzymatic kinetics of RNase A. In the middle of this chimeric substrate there lied one uracil nucleotide, with deoxyribonucleotides flanking this uracil nucleotide. One end of the oligonucleotide was attached to a fluorophore and the other end was attached to a quencher (James D A and Woolley G A. A fluorescence-based assay for ribonuclease A activity. Anal. Biochem., 1998, 264:26-33). The flaw of this solution is that it can merely be used to study the activity of certain RNases that can cleave uracil nucleotide, and on the other hand, a sensitive fluorophotometer is needed.
In another solution, Kelemen et al reported another similar technique (Kelemen B R, Klink T A, Behlke M A, Eubanks S R, Leland P A and Raines R T. Hypersensitive substrate for ribonucleases. Nucleic Acids Res., 1999, 27:3696-3701). Same as the research solution of James, this solution can only detect RNases that can cleave uracil nucleotide and needs a fluorophotometer.
In another solution, Burke et al reported detecting the cleavage activity of RNase on a synthetic substrate by utilizing fluorescence polarization technique (Burke T J, Bolger R E, Checovich W J and Tompson D V. Method and kit for detecting nucleic acid cleavage utilizing a covalently attached fluorescent tag. U.S. Pat. No. 5,786,139, 1998). At present, commercial kits based on this technique are available in the market (PanVera Corporation Catalog 2000, Section 3.10. 545 Science Drive, Madison, Wis. 53711). Wilson et al further improved this technique and realized the goal of real-time detection (Wilson G M, Lu H, Sun H, Kennedy A and Brewer G. A fluorescence-based assay for 3′→5′ exoribonucleases: potential applications to the study of mRNA decay. RNA, 2000, 6:458-464). However, this technique needs to use a fluorescence polarimeter that an ordinary laboratory does not have, impacting its extensive use.
Another commercial RNase activity detection kit is supplied by PanVera Corporation (PanVera Corporation Catalog 2000, Section 3.10. 545 Science Drive, Madison, Wis. 53711). Comparing with the fluorescence polarization solution, this technique has lower sensitivity.
Another commercial RNase activity detection kit is supplied by Ambion, Inc. This solution is based on an RNA substrate labeled by biotin, and detects RNase activity through common chromogenic reaction (Ambion, Inc. Catalog 1999, p104. 2130 Woodward Street, Austin, Tex. 78744.). Without RNase, the substrate can remain intact and turns blue in the chromogenic reaction; if the substrate is degraded by RNase in a sample, no blue reaction can be formed. This solution is labor intensive and is not suitable for high-throughput applications.
Another commercial RNase activity detection kit analyzes substrate degradation through gel electrophoresis (Mo Bio, Web Catalog, 2000). This solution involves multi-step reactions with heavy workload and is not suitable for routine detection.
To summarize, although the existing detection methods described above have made some progress in increasing sensitivity and accuracy for RNase activity detection, they all have some defects and are not suitable for being used as a universal means for detecting RNase activity in samples with high sensitivity. Related technical solutions still have much room for improvement.